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NTP Range-Finding Report on Immunotoxicity of Chloroform in Female B6C3F1 Mice (CASRN: 67-66-3)


The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program (NTP). The findings have not been peer reviewed and were not evaluated in accordance with the levels of evidence criteria established by the NTP on March 2009 (see The findings and conclusions for this study should not be construed to represent the views of the NTP or the US Government.

NTP Report Number: IMM98002


Disinfection byproducts (DBPs) are contaminants found in drinking water. DBPs are formed as by-products as a result of the chlorination/ozonization process used to purify water to acceptable drinking water standards. The potential effects of drinking water contaminants to affect adversely the immune system is a concern of both the Environmental Protection Agency (EPA) and the National Institute of Environmental Health Sciences (NIEHS). Several drinking water DBPs have been identified and selected for evaluation of their potential effects on the immune system in a joint project between the EPA and the NIEHS.

Trihalomethanes (THM) are volatile halogenated hydrocarbons generated by chlorination used in water treatment and disinfection. In most cases, chloroform (CHF) is the most abundant THM presented in treated waters. CHF has been classified as probable human carcinogen because high doses of CHF induce tumor formation in laboratory animals.

The National Toxicology Program (NTP) requested that a dose range-finding study be performed to establish the potential effects of CHF on the immune system and to determine doses that could be used in a full immunotoxicology study. These studies were conducted in female B6C3F1 mice. The animals were exposed to CHF based on the concentration of the test substance in the drinking water. Five CHF concentrations of 2.5, 10, 25, 100 and 250 ppm for 28 days were utilized. CHF solutions were prepared by the NTP in the National Institute of Environmental Health Sciences, and shipped to our facility. Water bottles were changed and filled twice per week. The in-life phase of these studies was carried out between August 10, 2000 and November 11, 2000.

For the baseline toxicology studies, exposure to CHF produced minimal changes in various immunological parameters.

CHF, when administered in the drinking water at doses from 2.5 to 250 ppm for 28 days, did not produce any signs of overt toxicity. With the exception of week 15, there was no significant difference in body weight between the exposed and control animals during the experimental period. Furthermore, over the total study period, there was no significant effect on body weight change. Taste aversion to CHF may have accounted for the initial significant decrease in water consumption at the two highest dose levels and decreased consumption of these groups throughout the study. At the time of sacrifice, no gross pathological lesions were observed in CHF-exposed animals. Exposure to CHF did not produce significant changes in the weights of thymus, liver, spleen, and kidneys. A decrease in lung weight was observed at the highest dose level, 250 ppm, and at the 25 ppm level. The hematocrit, MCV, MCH, MCHC, platelet count, percentage of reticulocytes, and total leukocyte count were unaffected by CHF. However, a slight decrease in the erythrocyte count (4%) and the concentration of hemoglobin (4%) was observed in the high dose group, and a decrease in neutrophils in the leukocyte differentials was observed. These toxicological changes demonstrate that CHF was being absorbed following exposure from drinking water. As in the toxicological parameters, exposure to CHF produced minimal effects in various immunological parameters. There were no biologically meaningful changes in the percentage and number of total B cells, T cells, CD4+ T cells, CD8+ T cells, natural killer cells and macrophages. Exposure to CHF did not produce a significant effect on the IgM antibody-forming cell responses to sheep red blood cells or serum IgM antibody titer. The effect of CHF on the activity of spleen T cells and natural killer (NK) cells was evaluated using the one-way mixed leukocyte response (MLR) and cytotoxic assay of YAC-1 cells, respectively. There was no biologically meaningful alteration in MLR and NK activity after exposure to CHF.

In conclusion, CHF, when administered in the drinking water at doses from 2.5 to 250 ppm, produced minimal toxicological and immunotoxic effects in female B6C3F1 mice. Based on the results of the range-finding study, CHF is not likely to have immunotoxicological effects in humans.

Report Date: March 2006

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